1. Field of the Invention
This invention relates to an apparatus and method for molecularly distilling ("drying") fluids from biological tissue. The dried tissue can be stored or otherwise used for any intended purpose. Experimentation has shown that the apparatus and method of this invention have demonstrated utility with tissue of various sizes and configurations. The specific end use of the tissue being dried is not considered a limiting factor in this invention.
Although the phrase "tissue sample" (the term "tissue" is used interchangeably with the term "tissue sample") is used throughout this disclosure, the term should be understood to include any material composed of one or more cells, either individual or in complex with any matrix or in association with any chemical. The definition shall include any biological or organic material and any cellular subportion, product or by-product thereof. The definition of "tissue sample" should be understood to include without limitation sperm, eggs, embryos and blood components. The contemplated utility of the apparatus of this invention is not limited to specific types or sizes of tissue. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue" and "tissue samples" are used interchangeably and are not limiting on the uses to which the method of this invention can be placed.
Also included within the definition of "tissue" for purposes of this invention are certain defined acellular structures such as dermal layers of skin that have a cellular origin but are no longer characterized as cellular. The term "component particles" is sometimes used as a generic reference to subunits making up "tissue" and should be understood to refer to molecules, individual cells or other subunits of tissue.
In one preferred embodiment of this invention the apparatus is used in conjunction with the preparation of tissue for ultrastructural analysis. Specifically, it is difficult to interpret the results of tissue analysis while concomitantly assessing the extent of various artifacts produced during the tissue preparation process. It is thus essential that artifacts be avoided wherever possible. The term "artifact" refers to a product of artificial character due to an extraneous agency. Another problem results from physical shrinkage of the tissue sample itself when subjected to the extreme procedures extant in current dogma. In most currently used tissue preparation steps, tissue shrinkage is in the order of 10% to 20%. This shrinkage inevitably results in alteration of ultrastructure and massive rearrangement of intrastructural resolution. The net result of this is ultrastructural translation damage and inaccurate detail in descriptions via existing analytical procedures.
During the so-called "Golden Age of Morphology" the predominant underlying goal in qualitative and quantitative microscopy has been an aesthetically pleasing image. This goal is readily attainable with the fixation methods and apparatus which are currently available. However, it has become essential in certain contexts that the aesthetically pleasing image, which is produced by the preparation process, also yield a tissue sample which accurately reflects the true condition of tissue in the living organism, i.e., approaching the "living state." Magnification apparatus which are currently available for analytical use are technically more advanced than current tissue preparation techniques which have been previously employed. The method of this invention results in the preparation of tissue samples which are readily usable on known magnification and analytical apparatus. Therefore, when used in conjunction with known cryopreparation-apparatus and methods the drying apparatus of this invention can be used to prepare tissue in essentially the "living state."
The "preparation" of tissue should be understood to refer to preparation of tissue for analysis as well as the drying of tissue in anticipation of transplantation, modification, in vitro or in vivo cellular growth, fertilization, animated suspension or the more typical resin impregnation, setting, infiltration and analysis. The method of this invention can be used to prepare tissue for any medical or analytical procedure.
The apparatus used in the practice of this invention is to be distinguished from contemporary freeze-drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freeze drying. See, for example, U.S. Pat. No. 4,232,453. Although in certain freeze-drying the tissue or sample itself does not attain a temperature approaching that of liquid nitrogen.
The vacuum levels disclosed and used in the apparatus used in the practice of this invention cannot be achieved safely with prior art freeze drying equipment. Typical of previous methods for drawing vacuums in freeze drying methods and apparatus is the above-mentioned U.S. Pat. No. 4,232,453 which discloses the use of molecular sieves in glass containers. Molecular sieves in easily compromised containers cannot be used safely to create and maintain the required vacuum levels to achieve the partial pressures required for sublimation of water at the anticipated temperatures (-120.degree. C. or below) created by the apparatus of the disclosed invention.
Throughout this specification the terms "distillation" and "distillation drying" are used. For understood to refer to the removal of liquid or solid materials, typically in molecular form, from a crystalline lattice. The term is intended to include sublimation and other physical processes whereby liquids, solids or materials that are present in a transition state between liquid and solid are removed. The specific characterization of the materials being removed from samples by the "distillation" process of this invention depend at least in part on the surface physics by which molecules are present within the crystal lattice of the material to be removed. Typically, molecules are removed from the tissue at the saturation vapor pressure of the material to be removed.
2. The Prior Art
Apparatus and methods for drying biological tissue in the past have been somewhat conventional. For example, as explained above freeze drying has been a well known technique for preparing and drying certain materials in the past. Other techniques involving conventional ovens and other heating methods have been known and used for many years. However, the process and apparatus of this invention have broken through conventional technical barriers. Under optimal conditions, water is molecularly distilled from tissue, preventing the ultrastructural and morphological damage that has previously been thought to be inherent in tissue drying.
Similarly, it is essential that drying methods and apparatus develop concurrently with other medical technology, i.e., surgical transplant techniques, bioengineering and biogenetics. In short, drying is an essential intermediate step in evolving processes using or analyzing cells or tissue. If drying apparatus does not evolve then the thrust of medical technology into unexplained and unexplored medical arts will be blunted. The method of this invention represents a drying breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical technology.
The most common alternative to chemical fixation and organic solvent dehydration is freeze drying cryofixed samples. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Cryofixation results in a near-instantaneous arrest of cellular metabolism. Freeze drying results in a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to cryofixation freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freeze-drying techniques to known tissue preparation processes. Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. These disadvantages deal primarily with damage to the cell ultrastructure during drying.
This general topic is discussed in some detail together with other prior art methods in an article entitled Freezing and Drying of Biological Tissues for Electron Microscopy, by Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in Understanding the Artifact Problem in Freeze-Fracture Replication: A Review. The Royal Microscopical Society, (1982) at pp. 103-123.
The particular physical state that the tissue is in when subjected to the drying process of this invention is not a limiting factor. In certain applications where ultrastructural damage must be controlled and minimized a cryoprepared tissue sample is used. Other applications do not require the absence of ice crystals and the tissue may be dried from the frozen condition. In even other situations a "room temperature" tissue sample can be used in the apparatus and method of the invention. The specific starting material and the physical or physiological conditions of the starting material are not limiting factors. The only essential characteristic of the tissue for use in the process and apparatus of this invention is that a distillable liquid, usually water, be present within the chemical structure of the tissue sample.
Dehydration is an essential step in the preparation of biological tissue samples for storage and a step which oftentimes results in the destruction via reticulation of the infrastructure and ultrastructure of the tissue. Tissue cell destruction from dehydration not only impairs analysis by magnification apparatus but also adversely affects the functional characteristics and viability of tissue masses being used, i.e. transplanted.
In certain prior art drying techniques, the tissue sample had not been entirely solidified due to eutectic formation as the cellular fluid solutes were concentrated in bound water compartments. This transfer of solute occurs while the materials are in the fluid state when slow cooling is employed. When rapid cooling techniques are used, unique procedures, which are distinct from those characteristic of freeze-drying, must be employed in the dehydration step. Problems result from the fact that dehydration must take place (the water must be removed) in the solid rather than the liquid state, i.e., via sublimation.
Also in the prior art, an alternative method of dehydration is referred to as the freeze substitution approach and involves the removal of tissue water by substituting a solvent or solvent-fixative mixture for the solid phase water at -50.degree. C. to -80.degree. C. This introduces less severe solvent phase separation and chemical alteration artifacts to a tissue sample than past routine chemical fixation methodologies.
From a practical standpoint freeze-drying is complicated by the requirement that the tissue sample be warmed to increase the vapor pressure of the supercooled water and to allow sublimation to proceed in a reasonable period of time. The increased temperature, in addition to increasing vapor pressure, can produce a series of physical events leading to the expansion of ice crystals and concomitant damage to the ultrastructural morphology of the tissue sample. Many of the physical events which occur during the warming process have to do with transitions in the physical state of the water which is present. Changes which are typically encountered are glass transition, devitrification and crystallization with an ensuing series of crystal lattice configurations.
The apparatus and method of this invention, which have been used successfully, are sometimes referred to as stimulated molecular distillation. Stimulated molecular distillation refers to a process in which the amount of energy in the antibonding orbitals of surface molecules is elevated, enabling the molecules to escape to the gas phase and not be recaptured by the solid phase.